Studies on the Nature of the Centromeric Nucleosome

着丝粒是一种特殊的核小体，作为有丝分裂纺锤体的附着点，在细胞分裂中起着重要作用。它们被假定为标准的八聚体组蛋白复合物，其中一个组蛋白H3被着丝粒特异性变体cenP-A（或cenH 3）取代。我们最近的研究和其他人的研究支持这样的假设，即至少在细胞周期的某些部分，着丝粒采用四聚体形式。其他研究指出，可能的着丝粒重塑在细胞周期和这些发现相结合，激发我们的调查可能的变化，其组成在细胞周期的不同阶段。 为此，通过免疫沉淀从同步化细胞中分离着丝粒，并使用生物化学和分子生物学工具进行检查。此外，我们使用原子力显微镜在单分子尺度上观察和比较细胞周期中着丝粒的形状，高度和体积。 结果表明，着丝粒是一种动态的结构，在周期中由四聚体和八聚体交替出现。 特别是，着丝粒在大部分周期中保持四聚体，但在DNA复制的S期改变其组成。 这种转变可能开始于从G1期到S期的转变，在那里我们开始看到四聚体和八聚体的混合。这种转化的机制尚不清楚，但这些发现揭示了新的过程，这将有助于我们进一步理解指导细胞周期的机制。

Centromeres are specialized nucleosomes playing an instrumental role in cell division as mitotic spindle attachment points. They have been assumed to exist as the standard octameric histone complex with one of the histones, H3, replaced by the centromere-specific variant cenP-A (or cenH3). Our recent studies and others' support the hypothesis that, at least during some part of the cell cycle, the centromere adopts a tetrameric form. Other studies point to possible centromere remodeling during the cell cycle and those findings, combined, motivate our investigation into possible variations in its composition at different stages of the cell cycle. For that purpose, centromeres are isolated from synchronized cells by immuno-precipitation and examined using biochemical and molecular biology tools. In addition we use atomic force microscopy to visualize and compare, at single-molecule scale, centromeres for their shapes, heights and volumes across the cell cycle. The results demonstrate that the centromere is a rather dynamic structure alternating between tetramer and octamer during the cycle. In particular, the centromere remains a tetramer for most of the cycle but changes its composition during the S-phase when DNA is replicated. The transformation probably begins during the transition from G1 to S-phase where we start seeing a mix of tetramers and octamers. The mechanism of such transformation is unknown, but the findings reveal novel processes that will help to further our understanding of the mechanics guiding the cell cycle.